This project will test the hypothesis that an important selective pressure for tRNA sequence diversity is the need for different aa-tRNAs to function equivalently in translation. It is proposed that the sequence of each tRNA body has been adjusted to compensate for the differing properties of the esterified amino acid and the differing strengths of the codon-anticodon interaction. Thus, each tRNA achieves uniformity in translation by interacting with EF-Tu and the ribosome in a slightly different manner. Aim 1: Directed mutations in EF-Tu will be used to refine our understanding of how E:F-Tu achieves specificity for tRNA sequences by an indirect readout mechanism. These same mutations will be tested for their activity in translation. Aim 2: The initial experimental goal is to master and improve the kinetic and thermodynamic assays to evaluate the function of tRNAs in the pre-translocation steps of translation on E. coli ribosomes. Several new assays will be developed to critically test the existing kinetic mechanism. These assays will then be used to evaluate the function of a set of mutant and chemically modified variants of tRNA with the overall goal of showing how tRNA sequences are "tuned" to function in a uniform manner. Aim 3: Biochemical assays on elongating ribosomes will test whether the activity of a tRNA in the A site is affected by the identity of the tRNA in the P site. Aim 4: Single deoxynucleotides inserted into the tRNA anticodon or the mRNA codon which disrupt the stabilizing A minor interactions will be used to determine at what stage in the reaction pathway the A-minor interactions form and to evaluate whether the A-minor interactions contribute to "smoothing out" differences in the codon-anticodon strengths among tRNAs.